All living organisms utilise adenosine triphosphate (ATP) as a source of chemical energy and it is known to assay this using the ATP driven luciferase/luciferin reaction. Light generated by this enzymic reaction can be measured using a luminometer and related to the amount of ATP present. The usefulness of ATP as an index of microbial numbers has been known since the mid 1960's (see ATP Luminescence Rapid Methods in Microbiology (1989) editor Stanley et al.; Blackwell Scientific Publications, London, see pages 1-10); its main advantage being speed and sensitivity. Utilising this assay format simple samples can be analysed in a matter of minutes while complex ones routinely take only half an hour with a detection capability provided down to 10.sup.-12 mol/l ATP. There is however a need for methods which provide still further sensitivity when detecting microorganisms or their contents while retaining speed and ease of performance.
The present inventor has now determined that the speed and sensitivity of the ATP based method can be enhanced significantly by shifting the target of the assay from ATP to the enzyme which generates it, adenylate kinase. Adenylate kinase is an enzyme used by all organisms for the conversion of adenosine diphosphate (ADP) and phosphate to adenosine triphosphate (ATP). The targeting of this enzyme in preference to ATP, by using the preferred method, apparatus and kits of the invention, allows the detection of down to at least 10.sup.-20 moles of intracellular marker adenylate kinase.
It is known to assay adenylate kinase using the luciferase/luciferin system (see Brolin et al Journal of Biochemical and Biophysical Methods 1 (1979) 163-169) for the purpose of determining its activity in certain mammalian and plant tissues (Rodionova et al Fiziologiya Rastenii (1978) 25, 4, p731-734 for plants). The use of such assay system for the detection of microorganisms however has not been suggested and the advantages of doing such, ie. enhanced sensitivity so provided, have not been relevant to those studying the enzyme itself.
Although adenylate kinase is present in smaller quantities than ADP or ATP, its use as a biological marker for microorganisms provides enhanced sensitivity with a typical amplification available of 400,000 by measuring its presence through the ATP it produces; that is for every mole of enzyme present 400,000 moles of ADP are converted to ATP in a 10 minute incubation. Thus estimation of the enzyme by measuring the substrate or product of the reaction it catalyses provides for detection down to as low as 10.sup.-20 moles.